Takamiya Tomoko, Hosobuchi Saeko, Asai Kenji, Nakamura Eiji, Tomioka Keisuke, Kawase Makoto, Kakutani Tetsuji, Paterson Andrew H, Murakami Yasufumi, Okuizumi Hisato
Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Electrophoresis. 2006 Jul;27(14):2846-56. doi: 10.1002/elps.200500776.
Restriction landmark genome scanning (RLGS) is a 2-DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, NotI as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites directly. We employed isoschizomers, MspI and HpaII, that recognize the same sequence (CCGG) but have different methylation sensitivity. We carried out the RLGS analysis of Arabidopsis thaliana ecotype Columbia, and obtained a pair of spot patterns with MspI and HpaII. We detected 22 spots in both patterns. In comparison of them, 18% of the spots were polymorphic, which indicated the methylation of C(5m)CGG sites. Further analyses revealed an additional methylated site of NotI. Moreover, 52 and 54 restriction enzyme sites were also analyzed in two other ecotypes, Wassilewskija and Landsberg erecta, respectively. Consequently, 15% of the 52 common sites showed methylation polymorphism among the three ecotypes. The restriction sites analyzed in this study were located in or near genes, and contribute new data about the correlation between methylation status and gene expression. Therefore, this result strongly indicates that the improved RLGS method is readily applicable to practical analyses of methylation dynamics, and provides clues to the relationship between methylation and gene expression.
限制性内切酶切位点基因组扫描（RLGS）是一种对基因组DNA进行的二维分析方法，可呈现数千个位点。在传统的用于甲基化分析的RLGS方法中，我们使用甲基化敏感限制性内切酶NotI作为标记。然而，它无法区分甲基化多态性和序列多态性。在此，我们报告一种改进的RLGS方法，可直接检测甲基化位点。我们使用了同裂酶MspI和HpaII，它们识别相同序列（CCGG）但甲基化敏感性不同。我们对拟南芥生态型哥伦比亚进行了RLGS分析，获得了用MspI和HpaII处理后的一对斑点图谱。我们在两种图谱中均检测到22个斑点。对它们进行比较，18%的斑点具有多态性，这表明C（5m）CGG位点发生了甲基化。进一步分析揭示了NotI的另一个甲基化位点。此外，还分别对另外两种生态型瓦西列夫斯基和直立型兰茨贝格的52个和54个限制性酶切位点进行了分析。结果，在这三种生态型中，52个共有位点中有15%表现出甲基化多态性。本研究中分析的限制性酶切位点位于基因内部或附近，为甲基化状态与基因表达之间的相关性提供了新数据。因此，这一结果有力地表明，改进的RLGS方法很容易应用于甲基化动态的实际分析，并为甲基化与基因表达之间的关系提供了线索。
